Association of PIK3CA Mutation With Pathologic Complete Response and Outcome by Hormone Receptor Status and Intrinsic Subtype in Early-Stage ERBB2/HER2-Positive Breast Cancer

Key Points Question What is the association of PIK3CA mutations, response to therapy, and outcome by hormone receptor (HR) status and intrinsic subtype among patients with ERBB2/HER2-positive early breast cancer (EBC) treated in a clinical trial? Findings In this cohort study of 184 patients enrolled in the phase 3 trial CALGB 40601, PIK3CA mutations were associated with lower pathologic complete response rates to chemotherapy plus trastuzumab and independently worse event-free survival, owing to the HR-positive and luminal intrinsic subtypes. Meaning This study suggests that PIK3CA mutations, which are therapeutically targetable, are associated with response to therapy as well as outcome in ERBB2/HER2-positive EBC, particularly luminal disease.


Introduction
ERBB2/HER2 overexpression or amplification defines the clinical ERBB2/HER2-positive breast cancer (BC) subtype.The introduction in clinical practice of ERBB2/HER2-targeted therapies has changed the natural history of ERBB2/HER2-positive BC.A long history of clinical (neo)adjuvant studies investigated the use of dual ERBB2/HER2 blockade to increase pathologic complete response (pCR) rates and improve outcomes in ERBB2/HER2-positive early BC (EBC).Currently, neoadjuvant chemotherapy plus dual blockade (trastuzumab plus pertuzumab, for a total of 1 year) represents the standard of care for ERBB2/HER2-positive EBC; however, response to treatment and survival outcomes remain highly variable.
In 30% to 35% of ERBB2/HER2-positive BCs, the protein phosphatidylinositol 3-kinase (PI3K) pathway, involved in ERBB2/HER2 oncogene signaling, 1 may be altered, mostly in the PIK3CA gene (OMIM 164870), encoding the PI3K α-catalytic subunit. 2 In ERBB2/HER2-positive BC, different hotspot PIK3CA mutations (most frequently in exons 9 and 20 2 ) have been associated with ERBB2/ HER2-independent activation of the PI3K pathway and resistance to anti-ERBB2/HER2 agents. 1,3As new anti-ERBB2/HER2 drugs were developed, new predictive and prognostic biomarkers have been developed, including intrinsic molecular subtype (IMS), immune gene expression, 4 and tumorinfiltrating lymphocyte counts, 5 as the most promising to tailor treatment.7][8][9] The implication of PIK3CA mutations in treatment response heterogeneity, long-term outcomes, and interaction with IMS within ERBB2/HER2-positive EBC is still unknown.Here, we studied the prognostic implications of PIK3CA mutations by hormone receptor (HR) status and IMS in the neoadjuvant Cancer and Leukemia Group B (CALGB) 40601 trial.
Cancer and Leukemia Group B is now part of the Alliance for Clinical Trials in Oncology.

Methods
1][12] This phase 3 trial randomized patients with newly diagnosed, untreated stage II or III ERBB2/HER2-positive EBC from 318 US and Canadian study locations between January 1, 2008, and December 31, 2012, to neoadjuvant weekly paclitaxel with trastuzumab, lapatinib, or both.Anthracycline-based chemotherapy plus completion of 1 year of trastuzumab was recommended after surgery.ERBB2/ HER2 testing was locally performed; positivity was defined by the trial eligibility as 3+ by immunohistochemistry (or 2+ with gene amplification by fluorescence in situ hybridization with a ratio of Ն2.0).Hormone receptor status was defined by local laboratory standards following American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines. 13is analysis followed Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK) guideline criteria 14

JAMA Network Open | Oncology
PIK3CA Mutation in Early-Stage ERBB2/HER2-Positive Breast Cancer A total of 305 patients were enrolled in CALGB 40601 and underwent 4 pretreatment 16-gauge core biopsies for research purposes, of whom 184 had adequate samples for DNA and RNA sequencing (RNASeq) (eFigure 1 in Supplement 1) and serve as this correlative study cohort.Wholetranscriptome analyses by RNASeq and whole exome sequencing were performed as specified (eMethods in Supplement 1), and all data are at the Database of Genotypes and Phenotypes (phs001570.v3.p1) and Gene Expression Omnibus (GSE116335).For gene expression profiling, whole-transcriptome analyses by RNASeq were performed in the University of North Carolina (UNC) High-Throughput Sequencing Facility and analyzed by the UNC Lineberger Comprehensive Cancer Center Bioinformatics Core.
The ethnic categories in the CALGB 40601 clinical trial protocol were Hispanic or Latino, not Hispanic or Latino, and not available.The racial categories included in the protocol were Black or African American, White, and other (including American Indian or Alaska Native, Asian, Native Hawaiian or Other Pacific Islander, and not available).In our cohort, 8 patients classified as "other" did not have information on race available.The ethnic distribution for these 8 patients were as follows: 6 non-Hispanic, 1 Hispanic, and 1 not available.

Statistical Analysis
Statistical analysis was performed from March 23, 2022, to March 9, 2023.Tumor and patient characteristics were analyzed using descriptive statistics.Event-free survival (EFS) was calculated from surgery to first recurrence (locoregional or distant) or death from any cause, and the median follow-up was 9.1 years (IQR, 8.0-9.9 years), with the study clinical database frozen on June 10, 2021.
Standard of care treatment of ERBB2/HER2-positive EBC includes trastuzumab; the lapatinib-only group was investigational, found inferior in several studies including this one, and was closed early. 15,16Response to therapy and prognostic analyses of PIK3CA mutation were preplanned in CALGB 40601; these were performed both in the overall cohort and, in an exploratory analysis, only in those treated with trastuzumab-based therapy, excluding those treated in the lapatinib group, as has been done previously. 10Logistic and Cox proportional hazards regression analyses were used to associate PIK3CA mutations with outcomes.
Hazard ratios, odds ratios (ORs), and 95% CIs were calculated and reported.The Kaplan-Meier method estimated 9-year EFS.All statistical tests were 2-sided, with P < .05 as significance level and performed using R, version 4.1.3(R Project for Statistical Computing), and Python, version 3.6 (Python Software Foundation).
b Included American Indian or Alaska Native, Asian, Native Hawaiian or Other Pacific Islander, and not available.In our cohort, the 8 patients classified as "other" did not have information on race available.

Discussion
We investigated the association of PIK3CA mutations in ERBB2/HER2-positive EBC with pathologic response to therapy and outcome by IMS and HR status in the phase 3 trial CALGB 40601 and found that PIK3CA mutations were associated with lower pCR rates among trastuzumab-treated patients and were independently associated with poorer 9-year EFS owing to associations in HR-positive and luminal subtypes.Our finding of worse pCR rates among patients with PIK3CA-mutated tumors is concordant with other trials 3,6,17 and with a pooled analysis of 5 neoadjuvant trials using the same drugs as in CALGB 40601 (trastuzumab with or without lapatinib).

Figure 1 .
Figure 1.Pathologic Complete Response (pCR) Rate of by PIK3CA Mutation in the Intention-to-Treat (ITT) Population and Patients Receiving Trastuzumab-Based Therapy 75

Table 1 .
Clinicopathologic Characteristics of Patients by PIK3CA Mutation Status 3

Table 2 .
Multivariable Model for Event-Free Survival CONSORT Diagram of DNA/RNA CALGB 40601 Population Analysis eFigure 2. Distribution of Different Types of PIK3CA Mutations eFigure 3. Distribution of PIK3CA Mutations by HR Status (A) and Intrinsic Subtype (B) in the Analytic Population eFigure 4. Event-Free Survival by PIK3CA Mutation in the Overall Population (A) and Multivariable Model Stratified by Hormone Receptor Status (B) eFigure 5. Event-Free Survival by PIK3CA Mutation in Combined Luminal A and Luminal B Breast Cancer Subgroup (A) and Multivariable Model Figure 2. Event-Free Survival by PIK3CA Mutation and Hormone Receptor (HR) Status